WO2006079205A1 - Methode de cryoconservation de cellules et de tissus par apport liposomal de sucres afin d'ameliorer la viabilite post-decongelation - Google Patents

Methode de cryoconservation de cellules et de tissus par apport liposomal de sucres afin d'ameliorer la viabilite post-decongelation Download PDF

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Publication number
WO2006079205A1
WO2006079205A1 PCT/CA2006/000098 CA2006000098W WO2006079205A1 WO 2006079205 A1 WO2006079205 A1 WO 2006079205A1 CA 2006000098 W CA2006000098 W CA 2006000098W WO 2006079205 A1 WO2006079205 A1 WO 2006079205A1
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WO
WIPO (PCT)
Prior art keywords
cells
sugar
cell
trehalose
liposomes
Prior art date
Application number
PCT/CA2006/000098
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English (en)
Inventor
Jason P. Acker
Maria Gyongyossy-Issa
Original Assignee
Canadian Blood Services
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Canadian Blood Services filed Critical Canadian Blood Services
Priority to EP06701865A priority Critical patent/EP1841856A1/fr
Priority to CA002595638A priority patent/CA2595638A1/fr
Publication of WO2006079205A1 publication Critical patent/WO2006079205A1/fr

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Classifications

    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A01N1/0205—Chemical aspects
    • A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
    • A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents

Definitions

  • U . S . Patent 4 , 327 , 637 (Morano et al . ) describes a method of sizing liposomes by extrusion .
  • a method for cryopreserving cells to enhance post-thaw cell viability .
  • the method includes the steps of loading sugar into liposomes ; fusing the liposomes to cell membranes of cells that are to be cryopreserved, thereby delivering the sugar into the cells as a cryoprotectant ; cooling the cells to a predetermined nucleation temperature; nucleating extracellular ice ; and cooling the cells to a temperature lower than the predetermined nucleation temperature .
  • Figure 2 is a bar graph plotting the recovery from O ' C and from -40 ° C of (i) red blood cells , (ii) red blood cells exposed to liposomes but containing no sugar and (iii) red blood cells exposed to liposomes containing 0.29M trehalose, as compared to 100% recovery from 22 'C (the control) ; and
  • a method for enhancing post-thaw viability of cryopreserved cells and tissues includes the step of loading sugar into liposomes (or "liposomal vesicles" ) .
  • the sugar-containing liposomes serve as delivery vehicles for delivering cryoprotectant sugar (s) into cells that are to be cryopreserved .
  • This method of cryopreserving cells enhances post -thaw cell viability .
  • the sugar utilized for liposomal delivery into the cells is selected from the group consisting of trehalose, sucrose, raffinose, and stachyose .
  • the sugar has a concentration between 0.05M and 0.50M .
  • trehalose is used in a concentration of 0.29M, although it should be understood that other sugars in similar or different concentrations can be used to work the present invention .
  • a concentration of 0.29M is - preferred because this concentration is iso- osmotic with normal saline and normal solutions that are typically used for cell suspensions .
  • the concentration of trehalose (TRE) can be varied provided there is salt present to offset a lower TRE concentration . While the range of 0.05M-0.5OM mentioned above is generally applicable to most monosaccharides and disaccharides , the optimal concentration for a given sugar will vary with a number of factors such as the molecular weight of the sugar. For example, raffinose may be protective at 100 mM ⁇ versus the 200 mM commonly used for trehalose .
  • sucrose or "sugar (s) " should be interpreted as including either a single type of sugar, e. g. trehalose, or a combination of sugars , such as for example a combination of sugars selected from the group consisting of trehalose, sucrose, raffinose, and stachyose .
  • the sugar (s) be dissolved in a solution having a physiologic osmolality, conductivity and pH .
  • Characterization of the final liposomal product is important for controlled, reproducible and effective liposomal delivery of cryoprotective intracellular disaccharides into mammalian cells for applications in biopreservation.
  • a number of techniques are used to characterize the liposomes , including fluorometry which enables one to measure the total mean fluorescence of ⁇ liposomes .
  • Mean fluorescence intensity increases relative to the [5 (6) -CF] in tile loading buffer, indicating controlled encapsulation of the dye .
  • Transmission electron microscopy can be also used to validate the size and to visually assess the morphology of the final liposome product .
  • the size of the liposomes also can be determined using dynamic light scattering .
  • a spectrophotometry assay of the phosphate content can estimate the total lipid concentration of the vesicle preparation, which allows for calculated and reproducible control of the liposome/cell ratio .
  • Figure 3 is a bar graph plotting the recovery from 22 °C, 0 ° C, -5 ° C, -10 ° C, -20 ° C, -30 ° C and -4O 0 C of (i) human platelets in buffer, (ii) human platelets in buffer exposed to liposomes without sugar, and (iii) human platelets in buffer exposed to liposomes containing 0.29M trehalose .
  • Figure 3 illustrates that post-thaw viability of platelets is enhanced by exposing the platelets to trehalose- containing liposomes .
  • the methods described in the present application satisfy a long-felt need in the ar,t of cryopreservation, particularly for clinical and commercial cell and tissue banking.

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  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Dentistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Environmental Sciences (AREA)
  • Medicinal Preparation (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne une méthode de cryoconservation de cellules impliquant l'apport liposomal de sucre(s) intracellulaire(s), tel que le tréhalose, le sucrose, le raffinose, la stachyose et des combinaisons de ces derniers, dans des cellules et des tissus, tels que des globules rouges, afin d'améliorer la viabilité post-décongélation. Ladite méthode permet l'apport rapide et aisé de molécules protectrices dans des cellules, ce qui simplifie ainsi considérablement la préparation de cellules pour la cryoconservation. De plus, étant donné que des concentrations beaucoup plus faibles de protecteur intracellulaire sont utilisées, la méthode permet la transfusion des globules rouges contenant le sucre intracellulaire apporté par voie liposomale à un patient immédiatement après la décongélation sans avoir à tout d'abord éliminer un quelconque sucre cryoprotecteur.
PCT/CA2006/000098 2005-01-28 2006-01-26 Methode de cryoconservation de cellules et de tissus par apport liposomal de sucres afin d'ameliorer la viabilite post-decongelation WO2006079205A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
EP06701865A EP1841856A1 (fr) 2005-01-28 2006-01-26 Methode de cryoconservation de cellules et de tissus par apport liposomal de sucres afin d'ameliorer la viabilite post-decongelation
CA002595638A CA2595638A1 (fr) 2005-01-28 2006-01-26 Methode de cryoconservation de cellules et de tissus par apport liposomal de sucres afin d'ameliorer la viabilite post-decongelation

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US64740305P 2005-01-28 2005-01-28
US60/647,403 2005-01-28

Publications (1)

Publication Number Publication Date
WO2006079205A1 true WO2006079205A1 (fr) 2006-08-03

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Family Applications (1)

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PCT/CA2006/000098 WO2006079205A1 (fr) 2005-01-28 2006-01-26 Methode de cryoconservation de cellules et de tissus par apport liposomal de sucres afin d'ameliorer la viabilite post-decongelation

Country Status (4)

Country Link
US (1) US20060188867A1 (fr)
EP (1) EP1841856A1 (fr)
CA (1) CA2595638A1 (fr)
WO (1) WO2006079205A1 (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008037481A2 (fr) * 2006-09-27 2008-04-03 Paul-Ehrlich-Institut Bundesamt Für Sera Und Impfstoffe Procédé de production amélioré de sang congelé ou de cellules sanguines congelées pour dosages biologiques
WO2013138239A1 (fr) 2012-03-14 2013-09-19 Membrane Protective Technologies, Inc. Systèmes et substances pour la cryoconservation de cellules viables
WO2016069173A3 (fr) * 2014-09-29 2016-07-28 Cook General Biotechnology Llc Utilisations du tréhalose dans des suspensions cellulaires
WO2024110457A1 (fr) * 2022-11-21 2024-05-30 Fraunhofer-Gesellschaft Zur Foerderung Der Angewandten Forschung E. V. Identification de cryoprotecteurs pour la cryoconservation de cellules et d'agrégats cellulaires

Families Citing this family (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ES2648139T3 (es) * 2010-02-19 2017-12-28 Cadila Pharmaceuticals Ltd. Una composición farmacéutica de células muertas con inmunogenicidad sustancialmente retenida
US20130331353A1 (en) * 2012-06-07 2013-12-12 Union College Cell-permeable variants of trehalose and methods for the protection of living cells
CN106062205A (zh) 2013-12-20 2016-10-26 基本药品有限责任公司 细胞培养基
US10448631B2 (en) 2015-09-22 2019-10-22 East Carolina University Cryopreservation using sucralose
JPWO2020066991A1 (ja) * 2018-09-28 2021-08-30 株式会社大塚製薬工場 アカルボース又はスタキオースを含む哺乳動物細胞保存用液
WO2020242970A1 (fr) * 2019-05-24 2020-12-03 East Carolina University Conservation à l'aide de sucres
CN111588648A (zh) * 2020-05-22 2020-08-28 广东创美抗衰老研究有限公司 自噬焕颜抗衰霜
WO2024024892A1 (fr) * 2022-07-28 2024-02-01 公立大学法人山陽小野田市立山口東京理科大学 Composition pour cryoconservation, procédé de cryoconservation et cellule congelée ou tissu biologique

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WO2000015032A1 (fr) * 1998-09-11 2000-03-23 The General Hospital Corporation Doing Business As Massachusetts General Hospital Poration controlee reversible pour la conservation de matieres vivantes
WO2001087062A2 (fr) * 2000-05-16 2001-11-22 The General Hospital Corporation Micro-injection de cytoprotecteurs pour la conservation des cellules
WO2004011616A2 (fr) * 2002-07-26 2004-02-05 The General Hospital Corporation Systemes et procede de conservation des cellules

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US4963362A (en) * 1987-08-07 1990-10-16 Regents Of The University Of Minnesota Freeze-dried liposome mixture containing cyclosporin
US6372720B1 (en) * 1998-02-05 2002-04-16 Kenneth J. Longmuir Liposome fusion and delivery vehicle

Patent Citations (3)

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Publication number Priority date Publication date Assignee Title
WO2000015032A1 (fr) * 1998-09-11 2000-03-23 The General Hospital Corporation Doing Business As Massachusetts General Hospital Poration controlee reversible pour la conservation de matieres vivantes
WO2001087062A2 (fr) * 2000-05-16 2001-11-22 The General Hospital Corporation Micro-injection de cytoprotecteurs pour la conservation des cellules
WO2004011616A2 (fr) * 2002-07-26 2004-02-05 The General Hospital Corporation Systemes et procede de conservation des cellules

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Title
BUCHANAN S.S. ET AL.: "Cryopreservation of Stem Cells Using Trehalose: Evaluation of the Method Using a Human Hematopoietic Cell Line", STEM CELLS DEV., vol. 13, no. 3, January 2004 (2004-01-01), pages 295 - 305, XP008120063 *
CONSTANTINESCU I. ET AL.: "Liposomes and Blood Cells: A Flow Cytometric Study", ARTIF. CELLS BLOOD SUBSTIT. IMMOBIL. BIOTECHNOL., vol. 31, no. 4, November 2003 (2003-11-01), pages 395 - 424, XP008120064 *
EROGLU A. ET AL.: "Intracellular trehalose improves the survival of cryopreserved mammalian cells", NAT. BIOTECHNOL., vol. 18, no. 2, February 2000 (2000-02-01), pages 163 - 167, XP008119934 *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008037481A2 (fr) * 2006-09-27 2008-04-03 Paul-Ehrlich-Institut Bundesamt Für Sera Und Impfstoffe Procédé de production amélioré de sang congelé ou de cellules sanguines congelées pour dosages biologiques
EP1908346A1 (fr) * 2006-09-27 2008-04-09 Paul-Ehrlich-Institut Bundesamt für Sera und Impfstoffe Procédé de préparation de sang ou de cellules sanguines congelés pour d'analyses biologiques
WO2008037481A3 (fr) * 2006-09-27 2008-10-09 Paul Ehrlich Inst Bundesamt Fu Procédé de production amélioré de sang congelé ou de cellules sanguines congelées pour dosages biologiques
WO2013138239A1 (fr) 2012-03-14 2013-09-19 Membrane Protective Technologies, Inc. Systèmes et substances pour la cryoconservation de cellules viables
EP2825635A4 (fr) * 2012-03-14 2015-09-23 Membrane Protective Technologies Inc Systèmes et substances pour la cryoconservation de cellules viables
US11028368B2 (en) 2012-03-14 2021-06-08 Membrane Protective Technologies, Inc. System and substances for cryopreservation of viable cells
WO2016069173A3 (fr) * 2014-09-29 2016-07-28 Cook General Biotechnology Llc Utilisations du tréhalose dans des suspensions cellulaires
WO2024110457A1 (fr) * 2022-11-21 2024-05-30 Fraunhofer-Gesellschaft Zur Foerderung Der Angewandten Forschung E. V. Identification de cryoprotecteurs pour la cryoconservation de cellules et d'agrégats cellulaires

Also Published As

Publication number Publication date
EP1841856A1 (fr) 2007-10-10
CA2595638A1 (fr) 2006-08-03
US20060188867A1 (en) 2006-08-24

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